Initiation of HIV-1 reverse transcription occurs at the primer binding site (PBS), that is complementary to the 3' terminal nucleotides of cellular tRNALyU. Studies from this laboratory during the last funding period have demonstrated that mutating the PBS to be complementary to alternative tRNAs resulted in viruses that could utilize these tRNAs to initiate reverse transcription. All of these viruses reverted to utilize tRNALys'3 after a short term of in vitro culture. Regions upstream of the PBS in U5, designated as the A-loop, have been shown to interact with the tRNA used for initiation; mutations in the A-loop that are complementary to the anticodon region, in addition to a PBS that is complementary to 3' terminal nucleotides, will allow the virus in some instances to stably utilize tRNAs other than tRNA ys'3 for replication. Using this information, in the last funding period, we have developed and characterized unique HIV-1 that utilize alternative tRNAs for initiation of reverse transcription. A novel complementation system was developed using HIV-1 modified to be infectious only if co-transfected with yeast tRNAphe. Using this system, we have determined that the important features of the tRNAphc that are required for selection and use in HIV-1 reverse transcription. This extension seeks to continue these studies with the following Specific Aims: Specific Aim 1: To continue to characterize the selection process for the tRNA necessary for HIV replication. Experiments are proposed to determine if aminoacylated tRNA can be selected for use in HIV reverse transcription and delineation of the step during tRNA biosynthesis that the tRNA is selected for use in reverse transcription. Specific 2: To delineate at the molecular level the interactions between tRNA-U5-PBS required for HIV replication. Experiments are described to determine how HIV-1 balances whether the viral RNA genome will be used for translation or encapsidation. Studies are proposed to determine if the tRNA interacts with the viral RNA genome prior to release and where in the cell this interaction occurs. Specific Aim 3: How does intracellular availability of tRNA influence HIV replication? We will determine if the low level of replication in PBMCs of some HIV-1 that utilize alternative tRNAs correlates with the tRNA availability. Using a novel system that was developed in this laboratory, we will ascertain whether viral genomes that encode the cognate tRNA complementary to the PBS replicate better in PBMCs than the wild type virus. Additional studies will examine whether the tRNA gene encoded by the viral genome is stable following replication. The results of our studies will provide us with essential information on the tRNA selection process HIV uses for initiation of reverse transcription that will lead to further development of new therapeutics to interrupt this process and facilitate the use of HIV-1 as a gene therapy vector.